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Tcf7l2/Tcf4 Transcriptional Repressor Function Requires HDAC Activity in the Developing Vertebrate CNS

View Article: PubMed Central - PubMed

ABSTRACT

The generation of functionally distinct neuronal subtypes within the vertebrate central nervous system (CNS) requires the precise regulation of progenitor gene expression in specific neuronal territories during early embryogenesis. Accumulating evidence has implicated histone deacetylase (HDAC) proteins in cell specification, proliferation, and differentiation in diverse embryonic and adult tissues. However, although HDAC proteins have shown to be expressed in the developing vertebrate neural tube, their specific role in CNS neural progenitor fate specification remains unclear. Prior work from our lab showed that the Tcf7l2/Tcf4 transcription factor plays a key role in ventral progenitor lineage segregation by differential repression of two key specification factors, Nkx2.2 and Olig2. In this study, we found that administration of HDAC inhibitors (Valproic Acid (VPA), Trichostatin-A (TSA), or sodium butyrate) in chick embryos in ovo disrupted normal progenitor gene segregation in the developing neural tube, indicating that HDAC activity is required for this process. Further, using functional and pharmacological approaches in vivo, we found that HDAC activity is required for the differential repression of Nkx2.2 and Olig2 by Tcf7l2/Tcf4. Finally, using dominant-negative functional assays, we provide evidence that Tcf7l2/Tcf4 repression also requires Gro/TLE/Grg co-repressor factors. Together, our data support a model where the transcriptional repressor activity of Tcf7l2/Tcf4 involves functional interactions with both HDAC and Gro/TLE/Grg co-factors at specific target gene regulatory elements in the developing neural tube, and that this activity is required for the proper segregation of the Nkx2.2 (p3) and Olig2 (pMN) expressing cells from a common progenitor pool.

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The histone deacetylase (HDAC) inhibitor VPA disrupts ventral progenitor boundaries.(A-F) Sections taken from E3 embryos treated with the indicated dilutions of VPA at E2 and stained for markers (Olig2, Nkx2.2, and FoxA2) that identify the three normally distinct ventral progenitor domains in the spinal cord. Inset schematic at bottom indicates the three progenitor domain boundaries analyzed and the corresponding markers used. pMN = motoneurons progenitor domain, p3 = V3 interneuron progenitor domain, FP = floor plate domain. Note that the number of double-labeled cells in both boundary regions increases significantly with increasing VPA concentrations (arrowheads) (A, D) controls; (B, E) VPA at 1mM; (C, F) VPA at 100mM). (G) Quantification of the results in A-F. *p<0.001.
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pone.0163267.g001: The histone deacetylase (HDAC) inhibitor VPA disrupts ventral progenitor boundaries.(A-F) Sections taken from E3 embryos treated with the indicated dilutions of VPA at E2 and stained for markers (Olig2, Nkx2.2, and FoxA2) that identify the three normally distinct ventral progenitor domains in the spinal cord. Inset schematic at bottom indicates the three progenitor domain boundaries analyzed and the corresponding markers used. pMN = motoneurons progenitor domain, p3 = V3 interneuron progenitor domain, FP = floor plate domain. Note that the number of double-labeled cells in both boundary regions increases significantly with increasing VPA concentrations (arrowheads) (A, D) controls; (B, E) VPA at 1mM; (C, F) VPA at 100mM). (G) Quantification of the results in A-F. *p<0.001.

Mentions: To test this, we first assayed whether HDAC activity was necessary for ventral progenitor patterning by examining Nkx2.2 and Olig2 expression after blocking endogenous HDAC activity in the spinal cord with Valproic Acid (VPA) [16], since prior studies have shown that numerous Class I HDACs are expressed in the developing chicken spinal cord [17]. Consistent with this, we found that the normally sharp boundary between motoneuron and V3 subclass interneuron progenitor domains (termed the “pMN” and “p3”, respectively) was progressively disrupted at increasing VPA concentrations 26 hours after treatment at E2, with many cells co-expressing Olig2 and Nkx2.2 and mixing into each domain (Fig 1A–1C and 1G). Similar results were obtained using 2 additional HDAC inhibitors, Trichostatin A and Sodium Butyrate (S1 Fig). These results are consistent with the idea that endogenous Nkx2.2 cannot repress Olig2 in the p3 domain, which occurs via a Gro/TLE/Grg-dependent mechanism [5], under conditions of attenuated or blocked endogenous HDAC function. In support of this, repression of Olig2 in the pMN domain by electroporation (EP) of Nkx2.2 was also blocked by VPA (S2 Fig). Additionally, the number of cells co-expressing Nkx2.2 and FoxA2 (marking floorplate (FP) cells) increased with VPA dosage, indicating a similar HDAC-activity dependence of cross-repression at the p3/FP boundary (Fig 1D–1G). HDAC inhibition did not affect progenitor proliferation or neural differentiation rates, as judged by examining mitotic (pHH3) and pan-neural post-mitotic marker protein (NeuN) expression in VPA treated embryos (data not shown). These data indicate that normal HDAC activity is required for the HD-mediated cross repression that refines the pMN/p3/FP domain boundaries in the developing neural tube.


Tcf7l2/Tcf4 Transcriptional Repressor Function Requires HDAC Activity in the Developing Vertebrate CNS
The histone deacetylase (HDAC) inhibitor VPA disrupts ventral progenitor boundaries.(A-F) Sections taken from E3 embryos treated with the indicated dilutions of VPA at E2 and stained for markers (Olig2, Nkx2.2, and FoxA2) that identify the three normally distinct ventral progenitor domains in the spinal cord. Inset schematic at bottom indicates the three progenitor domain boundaries analyzed and the corresponding markers used. pMN = motoneurons progenitor domain, p3 = V3 interneuron progenitor domain, FP = floor plate domain. Note that the number of double-labeled cells in both boundary regions increases significantly with increasing VPA concentrations (arrowheads) (A, D) controls; (B, E) VPA at 1mM; (C, F) VPA at 100mM). (G) Quantification of the results in A-F. *p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5036887&req=5

pone.0163267.g001: The histone deacetylase (HDAC) inhibitor VPA disrupts ventral progenitor boundaries.(A-F) Sections taken from E3 embryos treated with the indicated dilutions of VPA at E2 and stained for markers (Olig2, Nkx2.2, and FoxA2) that identify the three normally distinct ventral progenitor domains in the spinal cord. Inset schematic at bottom indicates the three progenitor domain boundaries analyzed and the corresponding markers used. pMN = motoneurons progenitor domain, p3 = V3 interneuron progenitor domain, FP = floor plate domain. Note that the number of double-labeled cells in both boundary regions increases significantly with increasing VPA concentrations (arrowheads) (A, D) controls; (B, E) VPA at 1mM; (C, F) VPA at 100mM). (G) Quantification of the results in A-F. *p<0.001.
Mentions: To test this, we first assayed whether HDAC activity was necessary for ventral progenitor patterning by examining Nkx2.2 and Olig2 expression after blocking endogenous HDAC activity in the spinal cord with Valproic Acid (VPA) [16], since prior studies have shown that numerous Class I HDACs are expressed in the developing chicken spinal cord [17]. Consistent with this, we found that the normally sharp boundary between motoneuron and V3 subclass interneuron progenitor domains (termed the “pMN” and “p3”, respectively) was progressively disrupted at increasing VPA concentrations 26 hours after treatment at E2, with many cells co-expressing Olig2 and Nkx2.2 and mixing into each domain (Fig 1A–1C and 1G). Similar results were obtained using 2 additional HDAC inhibitors, Trichostatin A and Sodium Butyrate (S1 Fig). These results are consistent with the idea that endogenous Nkx2.2 cannot repress Olig2 in the p3 domain, which occurs via a Gro/TLE/Grg-dependent mechanism [5], under conditions of attenuated or blocked endogenous HDAC function. In support of this, repression of Olig2 in the pMN domain by electroporation (EP) of Nkx2.2 was also blocked by VPA (S2 Fig). Additionally, the number of cells co-expressing Nkx2.2 and FoxA2 (marking floorplate (FP) cells) increased with VPA dosage, indicating a similar HDAC-activity dependence of cross-repression at the p3/FP boundary (Fig 1D–1G). HDAC inhibition did not affect progenitor proliferation or neural differentiation rates, as judged by examining mitotic (pHH3) and pan-neural post-mitotic marker protein (NeuN) expression in VPA treated embryos (data not shown). These data indicate that normal HDAC activity is required for the HD-mediated cross repression that refines the pMN/p3/FP domain boundaries in the developing neural tube.

View Article: PubMed Central - PubMed

ABSTRACT

The generation of functionally distinct neuronal subtypes within the vertebrate central nervous system (CNS) requires the precise regulation of progenitor gene expression in specific neuronal territories during early embryogenesis. Accumulating evidence has implicated histone deacetylase (HDAC) proteins in cell specification, proliferation, and differentiation in diverse embryonic and adult tissues. However, although HDAC proteins have shown to be expressed in the developing vertebrate neural tube, their specific role in CNS neural progenitor fate specification remains unclear. Prior work from our lab showed that the Tcf7l2/Tcf4 transcription factor plays a key role in ventral progenitor lineage segregation by differential repression of two key specification factors, Nkx2.2 and Olig2. In this study, we found that administration of HDAC inhibitors (Valproic Acid (VPA), Trichostatin-A (TSA), or sodium butyrate) in chick embryos in ovo disrupted normal progenitor gene segregation in the developing neural tube, indicating that HDAC activity is required for this process. Further, using functional and pharmacological approaches in vivo, we found that HDAC activity is required for the differential repression of Nkx2.2 and Olig2 by Tcf7l2/Tcf4. Finally, using dominant-negative functional assays, we provide evidence that Tcf7l2/Tcf4 repression also requires Gro/TLE/Grg co-repressor factors. Together, our data support a model where the transcriptional repressor activity of Tcf7l2/Tcf4 involves functional interactions with both HDAC and Gro/TLE/Grg co-factors at specific target gene regulatory elements in the developing neural tube, and that this activity is required for the proper segregation of the Nkx2.2 (p3) and Olig2 (pMN) expressing cells from a common progenitor pool.

No MeSH data available.


Related in: MedlinePlus